This was the initial year for this project, using biochemical methods to study conformational changes in ClC-type chloride channel proteins. The ClC family of chloride-conducting ion channels is involved in a host of biological processes; these channels maintain the resting membrane potential in skeletal muscle, modulate excitability in central neurons, and are involved in the homeostasis of pH in a variety of intracellular compartments. Despite their physiological importance, the mechanisms by which these channels function are poorly understood. We are attempting to understand the functional properties of these proteins by examining the function of several family members, including both eukaryotic and prokaryotic homologs. For the eukaryotic proteins, we are using combinations of site-directed mutagenesis and electrophysiological analysis of the channels. In contrast, for the prokaryotic channels, which we can heterologously express and purify in large quantities, we are using biochemical methods, including site-directed fluorescent labeling and radioactive flux measurements, to analyze the mechanisms of channel function. These two approaches provide complimentary results to improve our understanding of the ClC chloride channels at a biophysical level.